Antimicrobial and Antibiofilm Effects of Peptides from Venom of Social Wasp and Scorpion on Multidrug-Resistant Acinetobacter baumannii

Abstract

Intravascular stent infection is a rare complication with a high morbidity and high mortality; bacteria from the hospital environment form biofilms and are often multidrug-resistant (MDR). Antimicrobial peptides (AMPs) have been considered as alternatives to bacterial infection treatment. We analyzed the formation of the bacterial biofilm on the vascular stents and also tested the inhibition of this biofilm by AMPs to be used as treatment or coating. Antimicrobial activity and antibiofilm were tested with wasp (Agelaia-MPI, Polybia-MPII, Polydim-I) and scorpion (Con10 and NDBP5.8) AMPs against Acinetobacter baumannii clinical strains. A. baumannii formed a biofilm on the vascular stent. Agelaia-MPI and Polybia-MPII inhibited biofilm formation with bacterial cell wall degradation. Coating biofilms with polyethylene glycol (PEG 400) and Agelaia-MPI reduced 90% of A. baumannii adhesion on stents. The wasp AMPs Agelaia-MPI and Polybia-MPII had better action against MDR A. baumannii adherence and biofilm formation on vascular stents, preventing its formation and treating mature biofilm when compared to the other tested peptides.

Keywords: AMP; Acinetobacter baumannii; mastoparan; stent.

Figure 1

Acinetobacter baumannii biofilm formation. (A) The bacterial growth of Acinetobacter baumannii AB 02, AB 53, AB 72, and Escherichia coli (control) were incubated with LB + Glu for 24 h at 29 °C and the growth was determined by OD readings at 405 nm. (B) After this period, the presence of biofilms was evaluated using crystal violet dye staining. The bars represent the mean and standard deviations of triplicates. * Significant difference between biofilm formations by A. baumannii clinical isolates compared to E. coli (p < 0.05).

Figure 2

A. baumannii biofilm formations on fragments of cobalt–chromium vascular stents analyzed by SEM. (A) Coronary stent photography; the fragments used in the experiments were 6 mm long. (B) Architecture of the cobalt–chromium vascular stent, presenting two cells, connected by a link (arrow). (C) Stent structure enlargement of box in B. (D and E) Stent after incubation with A. baumannii AB 72 for 24 h under conditions for biofilm formation (arrow). (F) Protuberant bacterial accumulation, suggestive of initial biofilm formation (arrow head). Magnifications: (B) 40×; (C) and (D) 1000×; (E) and (F) 4000×.

Figure 3

Effect of the antimicrobial peptides (A) Agelaia-MPI, (B) Polybia-MPII, (C) Polydim-I, (D) Con10, and (E) NBDP 5.8 on the inhibition of adhesion of A. baumannii to 96-well polystyrene plates. The curves represent the bacterial growth of adhered cells on polystyrene plate by reading OD in the range of 405 nm. Results are reported as mean and standard deviations of triplicates. These results represent one of three independent experiments.

Figure 4

Effect of the antimicrobial peptides Agelaia-MPI and Polybia-MPII on the mature biofilm of A. baumannii on polystyrene plates. (A) Agelaia-MPI and (B) Polybia-MPII significantly inhibited mature biofilm at the lowest concentration of 6.25 μM. Results are reported as mean and standard deviations of triplicates. These results represent one of three independent experiments.

Figure 5

Effect of the antimicrobial peptides (A) Agelaia-MPI and (B) Polybia-MPII on A. baumannii-dispersed cells from biofilms on polystyrene wells. After washing the wells where biofilms had been previously formed and removing the non-adherent or poorly adhered bacteria, more culture medium was added with antimicrobial peptides and, 24 h later, bacteria present in the supernatant was measured. Results are reported as mean and standard deviations of triplicates. These results represent one of three independent experiments.

Figure 6

Role of the peptides Agelaia-MPI and Polybia-MPII on the inhibition of biofilm formation by A. baumannii on stents. (A and B) SEM evaluation of A. baumannii biofilms formed on stents. (C and D) Biofilm formation on stents by A. baumannii treated with Agelaia-MPI for 24 h. (E and F) Biofilm formation on stents by A. baumannii treated with Polybia-MPII for 24 h. Magnification of ×1000 (A, C and E); ×10,000 (B, D and F).

Figure 7

Coating of the vascular stent with Agelaia-MPI plus PEG prevented the adherence of A. baumannii. The vascular stent was left for 3 h in a PEG 400 solution containing 25 µM of Agelaia-MPI (stent + PEG + Agelaia−MPI) or not (stent + PEG). Coated stents were transferred to a new well containing fresh medium and then A. baummanii was added and incubated for 24 h. Uncoated stent was used as a control. At the end of incubation, wells were rinsed with fresh media and the stents were sonicated and the bacterial load adhered in each situation was determined. Results are reported as means and standard deviations of triplicates from one of three independent experiments. These results in all three independent experiments were similar, * p < 0.05 and ** p < 0.0001.

Figure 8

Effect of Agelaia-MPI and Polybia-MPII AMPs on the adherence and biofilm formation of Staphylococcus strains. (A) Agelaia-MPI activity against adhesion and (B) biofilm. Polybia-MPII activity against (C) adhesion and (D) biofilm. Results are expressed as means and standard deviations of triplicates. These results represent one of three independent experiments.